INTEGRATED DNA GENOTYPING AND SEQUENCING MICROSYSTEMS

DNA基因分型和测序的分析方法连接成从DNA源材料开始提取遗传信息的多步骤处理系统。在传统的方法中，处理系统与配套的液体处理装置和电泳分析设备一起对微升体积的反应样品进行处理。项目1将提供一个系统级的方法来评估集成的纳升体积组件和在项目项目中开发的制造技术。微加工组件的成功将由其作为自主多步骤加工系统的能力来衡量。项目1有四个具体目标：1)开发集成纳升级DNA分析所需的微流体。微流控元件将用于构建一个集成系统，可以执行和监测多个平行反应。还将讨论与微升体积源样本的接口。2)在集成系统中演示测序和基因分型反应。微流控装置将优化用于桑格测序或基因组扩增反应和变性凝胶电泳。系统级设备将执行和监测聚合酶反应、反应后处理和电泳带迁移，操作人员干预最少。3)在集成系统中展示高质量的基因组DNA测序。提高集成纳升-体积系统的分辨率，与现有微升级集成纳升-体积系统的分辨率相匹配；这将包括改进的荧光检测器（项目3）和更高分辨率的凝胶（项目2）。这一目标的成功完成将导致一种可以从模板DNA样本中获得500-1000 bp序列的设备。4)在集成设备中演示复杂的测序策略。从单个DNA样品中获得最大信息的复杂测序策略，如双向测序或同时限制性内切酶切，将被添加到集成测序设备中。质量控制、准确性和数据可重复性的问题也将被审查。

Analysis methods for DNA genotyping and sequencing are linked into multiple-step processing system that begin with DNA source material and extract genetic information. In conventional methods, the processing system operates on batches of microliter-volume reaction samples, together with matched-liquid handling devices and electrophoretic analysis equipment. Project 1 will provide a systems-level approach to evaluate the integrated nano-liter volume components and fabrication technologies developed in the Program Project. The success of microfabricated components will be measured by their ability to work as an autonomous multi-step processing system. Project 1 has four Specific Aims: 1) Develop the microfluidics necessary for integrated nanoliter-scale DNA analysis. Microfluidic components will be used to construct an integrated system that can perform and monitor multiple parallel reactions. An interface with microliter-volume source samples will also be addressed. 2) Demonstrate sequencing and genotyping reactions in an integrated system. Microfluidic devices will be optimized for use with Sanger sequencing or genomic amplification reactions and denaturing gel electrophoresis. The system-level device will perform and monitor polymerase reactions, post reaction treatment, and electrophoretic band migration, with minimal operator intervention. 3) Demonstrate high-quality genomic DNA sequencing in an integrated systems. The resolution of the integrated nanoliter-volume system will be improved to match the level of existing microliter-scale integrated nanoliter-volume system will be improved to match the level of existing microliter-scale technologies. This will include improved fluorescence detectors (with Project 3) and higher resolution gels (with Project 2). Successful completion of this aim will result in a device that can obtain 500-1000 bp of sequence from a sample of template DNA. 4) Demonstrate complex sequencing strategies in an integrated device. Complex sequencing strategies that obtain maximal information from a single DNA sample, such as bi-directional sequencing or simultaneous restriction digestion, will be added to the integrated sequencing device. Issues of quality control, accuracy, and data reproducibility will also be examined.